The sensation of pain due to injury or disease is carried from the periphery to the brain by a multi-neuronal pathway. The first part of this system comprises the primary nociceptive afferents that form synapses with secondary neurones in the dorsal horn of the spinal cord, or the nuclei of the cranial nerves. These synapses pass on the incoming information by the release of neurotransmitters and neuromodulators such as glutamate and substance P. These synapses are, therefore, possible sites for intervention to alleviate pain, indeed one of the modes of action of the opiate analgesics is to down-modulate neurotransmitter release at these synapses.
Unfortunately, the opiates have a number of limitations as drugs. Firstly, there are a number of chronic pain conditions for which the opiates are not effective. Secondly, the opiates have a number of side effects that are mediated both peripherally (constipation) and centrally (respiratory depression and euphoria) which present problems for long term use.
There is, therefore, a need for the development of new pharmaceuticals for the treatment of pain, particularly chronic pain.
One approach to this problem is the use of new agents containing fragments of clostridial neurotoxins (WO96/33273).
The clostridial neurotoxins are proteins with molecular masses of the order of 150 kDa. They are produced by various species of bacterium of the genus Clostridium, most importantly C. tetani and several strains of C. botulinum. There are at present eight different classes of the neurotoxins known: tetanus toxin, and botulinum neurotoxin in its serotypes A, B, C1, D, E, F and G, and they all share similar structures and modes of action. The clostridial neurotoxins are synthesised by the host bacterium as single polypeptides that are modified post-translationally to form two polypeptide chains joined together by a disulphide bond. The two chains are termed the heavy chain (H), which has a molecular mass of approximately 100 kDa, and the light chain (L), which has a molecular mass of approximately 50 kDa. Two distinct functions can be identified within the H-chain; binding and translocation. The carboxy-terminal half (HC) is involved in the high affinity, neurospecific binding of the toxin to cell surface acceptors, whilst the amino-terminal half (HN) is central to the translocation of the toxin into the neuronal cell. For botulinum neurotoxin type A these domains are considered to reside within amino acid residues 872-1296 for the HC, amino acid residues 449-871 for the HN and residues 1-448 for the LC. The minimal domains necessary for the activity of the light chain of clostridial toxins are described in J. Biol. Chem. Vol. 267, No. 21, July 1992, pages 14721-14729. The eight distinct neurotoxin light chains (L) are highly specific zinc-dependent endopeptidases which each hydrolyse different but specific peptide bonds in one of three substrate proteins, synaptobrevin, syntaxin or SNAP-25. These substrates are important components of the neurosecretory machinery. The hydrolytic activity of the clostridial toxins results in a prolonged muscular paralysis. The functions of all three identified domains are necessary for the toxic activity of the clostridial endopeptidases.
Some of the clostridial endopeptidases, most notably botulinum neurotoxin type A, have been used as pharmaceutical agents for the treatment of a range of muscle dystonias. The flaccid paralyzing action of the native botulinum toxins makes them appropriate for this use.
The use of fragments of clostridial neurotoxins for the desired purpose of analgesia is dependent on the invention of conjugates, or derivatives of these molecules, with a specific binding activity that will deliver the L-chain endopeptidase to the nociceptive afferent neurons in preference to other neurones in the relevant anatomical locus. Delivery of these conjugates includes binding to the cell surface, internalization via an endosomal compartment and translocation of the clostridial endopeptidase activity into the cytosol.
Targeting of extracellular species to specific intracellular locations following endocytosis involves an appreciation of a number of possible targeting strategies. It is understood that early endosomes are part of the key sorting mechanisms of the cell, routing species to late endosome (and onto lysosomes for degradation), recycling to the cell surface or to the Trans-Golgi Network. Intracellular routing determinants have been suggested that determine the pathway and final destination of particular species (Mellman, 1996, Annu. Rev. Cell Biol., 12, 575-625).
Current data suggests that translocation of native clostridial neurotoxins occurs from an acidic intracellular compartment, though the exact location and nature of the compartment is unknown (Montecucco & Schiavo, 1994, Mol. Micro. 13, 1-8). In patent WO96/33273 it is proposed that for an agent to be effective, the agent must target to an appropriate compartment for translocation of the toxin. As an example of specific intracellular targeting, internalization of the NGF-receptor is by specific endocytosis and retrograde routing (initiated by receptor-ligand complex), via acidic endosomes to the cell body, and an agent incorporating NGF is given in support of WO96/33273.